**6. Conclusions**

*Current Topics in Biochemical Engineering*

expression in the parent cell would be induced to deliver EVs enriched in the modified protein. The success of such set-up depends on the understanding of both

In one of the pioneering studies, certain sites on the tetraspanin CD63 have been chosen to allow the integration of fluorescent fusion proteins on the extraand intravesicular side of the exosomal membrane [105]. CD63-GFP fusions have proven valuable reporters in the elucidation of the role of immune synapse in secretion of exosomes from T-cells to APCs and their fusion with recipient cells [106], in determining differential uptake properties of different immune cell subsets for EVs originating from a cancer cell line [107] and in an *in vivo* study imaging the fate of

**5.3 Engineering of EV surface proteins for enhanced cell-type specific targeting: introduction of target specificity, stability and improved cargo delivery**

In the pioneering example of derivatization of a protein enriched in exosomal membrane to achieve specific targeting [109], the rabies virus glycoprotein (RVG) peptide fused with lysosome-associated membrane glycoprotein 2b (Lamp-2b) was used to target exosomes to the central nervous system in an *in vivo* mouse model. Immature dendritic cells-derived exosomes, enriched in an N-terminal fusion of an αv integrin-specific internalizing RGD-sequence containing peptide with this scaffold, could internalize efficiently into target-positive breast cancer cells [110]. The display of such constructs could be efficiently enhanced by introducing a protective glycosylation motive, which improved the surface expression of exosome-bound N-terminally fused peptides by preventing their acid-mediated proteolysis during endosomal passage and indeed led to a more efficient specific cellular uptake of exosomes engineered in this way [111]. Specific targeting of IL3-receptor overexpressing chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells has been shown for exosomes armed with a fusion protein of IL3 and Lamp2b and loaded with Imatinib, a tyrosine kinase inhibitor, and led to a the reduction of tumor size *in vivo* [112]. Exosomes engineered in this way achieved a higher abundance at the tumor site and were hence able to inhibit xenograft growth more efficiently than the active ingredient alone or than loaded control exosomes. Glycosylphosphatidylinositol (GPI)-mediated anchoring of the specific targeting unit was demonstrated to be a stable alternative to Lamp2b fusions. EVs expressing GPI-anchored nanobodies, specific to epidermal growth factor receptor (EGFR), displayed enhanced binding to the EGFR-overexpressing cancer cells. This, however, did not lead to an increased uptake, and it was suggested that in this particular biological system not only the affinity but also the density of the targeting ligand must be high enough to induce receptor clustering and subsequent internalization [113]. Exosomes derived from a HEK cell line transfected with a construct of platelet-derived growth factor (PDGF)-anchoring sequence and an EGFR-binding peptide were on the other hand efficient in targeting EGFRexpressing tumors *in vivo* and reducing their size with delivery of microRNA Let7 [114]. Enhanced uptake has been achieved for EVs enriched in a fusion protein of tetraspanin CD63 and stearylated octaarginine, a representative cell-penetrating

molecular biology and protein architecture of the targeted species.

EVs produced by breast cancer cell line in *nude* mice [108].

peptide, by their ability to induce active macropinocytosis [115].

The concept of enhancing the stability of the targeted exosomal surface protein intended for fusion with a targeting agent to allow a higher degree of versatility for their modification for an improved target recognition has raised interest in their engineering at the protein level. A significant increase in thermal stability has been achieved by introduction of additional disulfide bonds in the EC2 of tetraspanin CD81, with the best variant exhibiting a positive shift in the melting temperature

**114**

The tetraspanins, well established as the biomarker proteins of extracellular vesicles, have been addressed for increasing EV stability and improving their function as delivery vehicles, both by assigning them with target recognition properties and modulating their cargo transfer. From the current point of view, the complexity of the tetraspanin-mediated interactions and signaling networks formed in a cell is yet to be discerned. Tetraspanins are known to interact naturally with a plethora of cell surface-bound ligands, which results in potent biological effects conveyed through different pathways; however, systematic evaluation of the affinity of the association with the interaction partners would assist in prediction of the consequential cellular processes as well as in determining optimal choice of the tetraspanin targeted for modification. There are recent reports describing modified tetraspanins mediating both surface protein interactions and an intracellular fusion-mediated enzymatic activity, which underline the feasibility of engineering versatile functions into tetraspanin proteins as fusion partners. The structural details on tetraspanins modified in this way, as well as the read-out revealing their actual behavior in the foreseen role and the influence of such modifications on

the fate of an EV preparation, will pave the way into the design and production of EV-based reagents as therapeutics.
